L20- Cell cycle

Regulation of cell cycle proteins

• Phosphorylation (kinase vs phosphatases)

  • lots of diff kinases, key= CDKs

  • lots of diff phosphatases

• Ubiquitination (E3 ubiquitin ligase complexes) + proteasome

  • SCF, APC, Mdm2

• Transcriptional control (TR factors)

  • lots of key ones

    • E2F, Myc, p53, pRB

3 main check points in cell cycle

• G1/S check pt→ decision to replicate genome (entry into S)

• G2/M→ decision to divide into 2 cells (entry into M)

• Metaphase→ decision to proceed w/ genome division (completion of M)

• Diff cyclins regulate diff check pts, expression + degredation reg in a cyclical pattern

Cell cycle progression controlled through ____ and ____

• coordinated reg of gene expression= promoter regulation

• protein degradation (ubiquitination)

APC and SCF are

• key ubiq E3 ligases that Ub-tag proteins in cell cycle dependent manner

  • ex) they target proteins req for licensing replication origins to avoid re-licensing (2x)

How do we study the cell cycle

• flow sorting

• histology/immunofluorescence

• Live cell imaging + reporters

Flow sorting

• Cells stained w/ dye

• amount of DNA in each cell measured as they pass through laser

• Tells which phase of cell cycle each cell is in

  • G1= 1x DNA, S= bw 1 and 2x, G2/M= 2x DNA

Histology/immunofluroscne

• tissue/cell samples fixed and stained with antibodies that recognize cell cycle markers

• Fluorescent tags let you see which cells in specific stages under microscope (red/green)

• Purpose→ show pattern of cell cycle phases in tissues/individual cells

Live cell imaging + reporters

• Cells engineered to express fluorescent reporters that change during cell cycle

• Watch cells progress through cycle in real time

• ex) make fusion protein with NLS and NES, NLS is whats seen and stays in nucleus but if phosphry by CDK2→ hides NLS and NES exposed so protein leaves nucleus and shows in cytoplasm

• CDK activity changes in each stage so depending on how much protein seen in nucleus/cytosol can det which stage

How do mother cells affect their daughter cells?

• influence whether daughter goes to G0 or G1

• if mother cell undergoes stress, daughter cells dont divide (stress like DNA damage etc)

Miotic spindle

• responsible for chromosome movement during mitosis

• Microtubule growth increases rapidly during late prophase as spindle fibers form

  • attach to kinetochore region at centromere

Spindle assembly checkpt, what happens if not properly set up

• ensuring proper metpahase-anaphase transition

• Checks spindle properly set up

• IF spindle not properly set up can cause mitotic catastrophe= aneuplodies=cell death (hopefully)

Key players of spindle assembly checkpt

• Cohesin→ holds together sis chromatids

• Seperase→ degrades cohesin

• Securin→ inhibits seperase

• APC (anaphase prom complex)→ E3 ubiq ligase, targets securin

• Cdc20→ Activates APC

Spindle assembly checkpt if all is well then

• APC (E3 ubq ligase) degrades

  • securin, cyclin B (main mitotic cyclin)

• Seperase free to digest cohesins

  • releases sis chromatids

• Metaphase→ Anaphase transition

• cells exit mitosis

What is spindle assembly checkpt gatekeeping and how is this spindle assembly check pt regulated

• gatekeeps entry into anaphase UNTIL all microtubules are attached to their kinetochores

• Mad and Bub proteins regulate checkpt proteins

When kinetochores are NOT attached

1. Unattached kinetochore recruits Mad/Bub proteins

2. Mad binds Cdc20

3. Bub proteins join→ form the mitotic checkpoint complex (MCC)

4. This complex binds APC and inhibits it

Result→ APC cannot degrade securin, sister chromatids stay together→ NO ANAPHASE

When all microtubules attached to kinetochores

1. Complex falls apart, Mad2 can no longer bind Cdc20 and Bub proteins

2. Cdc20 now free

3. Cdc20 binds APC in a DIFFERENT spot→ activates APC

Result→ APC degrades securin→ seperase activated→ digests cohesin→ sister chromatids separate→ anaphase begins

G0 vs G1

• cell exited mitosis now either in G1/G0

• Determined based on the situation during previous cell cycle

  • normal situation= G1

  • Situation critical= G0

• If in G1, cell begins accumulating cyclins towards S phase entry

  • Major checkpt= restriction pt

  • pRB phosphorylation status

Restriction point checkpoint

• ensuring DNA replication doesnt occur prematurely

• Checks genome + envt OK to proceed

• Stakes→ If S phase entered when there’s DNA damage/ envt not favorable (not enough energy/resources) or some other stress→ genome instability (mutaations) or stressed daughter cells→ unstable cells

Key players of restriction pt checkpt

• pRB→ inhibits E2F

• E2F→ promotes expression of cell cycle factors

• p53→ master reg, stress sensor

• Cyclin dependent kinases CDKs→ Phosphorylate pRB to inactivate it

• Cyclins→ activate CDKs

• CDK inhibitors→ inhibit CDKs

blue=TFs

E2F proteins sit on promoters of genes involved in

• DNA repair: BRCA, RAD, MutS, etc

• DNA replication: DNA pol, PCNA< ORC etc

• Cell cycle factors: Mad, Bub, P53, Cdc2, Clyclin A, pRB etc

• Chromatin assembly, remdoelling, segregation etc

pRB-E2F vs CDK/cyclins vs p53/CDKi

• CDK + Cyclin form an active complex→ this complex phosphorylates retinoblastoma protein (pRB) → turns pRB OFF because now it has changed conformation so can’t bind E2F, allows expression of genes for S phase (DNA rep) bc RNA pol can transcribe→ S phase begins

  • Normally though unphosphorylated pRB binds and blocks E2F (TF) → no gene expression for S phase

• p53 turns on genes for CDKi→ CDKs get blocked → no phosphorylation of pRB→ pRB stays active so keeps blocking E2F→ no gene expression → Cell cycle stops before S phase

p53 responds to stress like DNA damage by inducing CDKi,→ preventing pRB phoshorylation→ blocking E2F driven transcription

CDK activation

• Cyclin + CDK→ form a complex

• CDK-Activating kinase (CAK) phosphorylates the CDK

• Cdc25 (phosphatase) removes the inhibitory phosphate→ CDK fully active

How CDKs are inhibited by INKs and CIP/KIPs

• CDK + Cyclin+ ATP→ active, phosphorylated substrate

• INK4 inhibitors→ binds CDK→ conformational change impacting a key active site loop→ inactivating the protein + prevents cyclin from interacting with CDK

  • allosteric inhibition

• Kip1 inhibitors→ binds CDK active site→ ATP cant bind→ enzyme inactive

  • competitive inhibition

p53 regulation (guardian of genome)

• Phosphorylation (ATM/ATR kinases)= activate

• Ubiquitination (MDM2)= block

• p53 activation→ cell cycle arrest/death

  • arrest (pause), or senescence (indef pause), or death (apoptosis)

How is p53 cell cycle arrest mediated

• p16INK4A, p15INKB, p19INK4D = block CDK4, 6

• CIP/KIP: p21Cip1, p27Kip1, p57Kip2 = block CDK4, 6,

p53 cell death mediated through

• PUMA→ block BCL-2